The field of this invention is arrays having associated oligonucleotides and/or polynucleotides, methods of producing such arrays, and uses thereof.
Arrays of binding agents, such as oligonucleotides and polynucleotides, have become an increasingly important tool in the biotechnology industry and related fields. These arrays, in which a plurality of binding agents are deposited onto a solid support surface in the form of an array or pattern, find use in a variety of applications, including drug screening, nucleic acid sequencing, mutation analysis, and the like. One important use of arrays is in the analysis of differential gene expression, where the expression of genes in different cells, normally a cell of interest and a control, is compared and any discrepancies in expression are identified. In such assays, the presence of discrepancies indicates a difference in the classes of genes expressed in the cells being compared.
In methods of differential gene expression, arrays find use by serving as a substrate with associated binding fragments such as oligonucleotides. Nucleic acid sequences are obtained from analogous cells, tissues or organs of a healthy and diseased organism, and hybridized to the immobilized set of binding fragments associated with the array. Differences between the resultant hybridization patterns are then detected and related to differences in gene expression in the two sources.
A variety of different array technologies have been developed in order to meet the growing need of the biotechnology industry. Despite the wide variety of array technologies currently in preparation or available on the market, there is a continued need to identify new array devices to meet the needs of specific applications. Of particular interest are arrays that provide increased binding affinity, because these allow the use of shorter binding fragments and fewer bound fragments can be used to obtain the results currently available with conventional technology. Also of interest is the development of an array capable of providing high throughput analysis of differential gene expression or identification of nucleic acids for diagnostic purposes, where the array itself is reusable. Such an array is needed for a number of reasons such as decreasing experimental variability, confirming results, and for decreasing costs of such analysis.
The present invention provides arrays having associated polymer sequences which are preferably oligonucleotide and/or polynucleotide polymers with modified structures (e.g., 1xe2x80x2, 2xe2x80x2, 3xe2x80x2, 5xe2x80x2 and/or modifying the ribose oxygen), methods of making such arrays, assays for using such arrays, and kits containing such arrays. The arrays of the present invention are attached to the substrate surface via a non-covalent linkage, e.g., a modified polynucleotide having a biotin group is attached to a substrate having a surface coated with avidin. The modifications described herein provide numerous advantages, including ease and efficiency of manufacturing probes with a higher binding affinity for complementary nucleic acids, acid resistance, and/or nuclease resistance.
The invention comprises an array device comprised of a support surface and polymer molecules bound to the support surface. The polymer molecules may be, but are preferably not naturally occurring oligonucleotides or polynucleotides, but rather are unique polymers having modified backbones with bases attached in the desired sequential positioning and the desired spacing between the bases. Because of the biotin-avidin interaction the manufacturing methodology of the invention can be readily adapted to produce arrays with any type of pre-made polymer. In a preferred embodiment a modified oligonucleotide is provided wherein the backbone is preferably modified to obtain improved results compared to natural oligonucleotides or polynucleotides including (1) higher binding affinity with RNA; (2) greater acid resistance; (3) greater resistance to enzymatic degradation; and/or (4) overall better performance and reusability.
In one embodiment, the modified associated oligonucleotide and/or polynucleotide polymers of the invention provide additional binding affinity with respect to corresponding, unmodified oligonucleotides having the same sequence. The binding affinity is preferably increased by a modification at the 2xe2x80x2 site of the sugar group, e.g., a 2xe2x80x2-F or a 2xe2x80x2-OR modification wherein R is a lower straight or branched change alkyl containing 1 to 6 carbons and is preferably 2xe2x80x2-O-methyl or 2xe2x80x2-methoxyethoxy. Alternatively or in combination, the binding affinity can be increased by modification in the 3xe2x80x2 linkage group, e.g., phosphoramidate linkages, or a modification replacing an oxygen in the phosphate linkage with a carbon.
Another aspect of the invention is a method of producing high density arrays whereby high density spots of avidin are placed on a surface. Polymer sequences are produced where the sequences are connected to biotin preferably via a linker. A plurality of identical sequences are then attached to a given avidin spot on the surface via the biotin-avidin binding. Different sequences are attached to other avidin spots thereby producing an array.
In another embodiment, the modified associated oligonucleotide and/or polynucleotide polymers of the array exhibit substantial acid resistance, allowing the arrays to be treated with low pH solutions. This allows the array to be exposed to low pH in order to remove any bound nucleic acids that are not modified, e.g., bound test nucleic acids.
It is thus an object of the present invention to provide arrays having associated chemically modified oligonucleotide and/or polynucleotide polymers characterized by substantial acid resistance. Such arrays may be exposed to low pH environments to facilitate clearance from the array of the test nucleic acids.
In yet another embodiment, the modified associated oligonucleotide and/or polynucleotide polymers of the array exhibit substantial resistance to nuclease degradation. These molecules preferably have an end-blocking group that confers nuclease resistance to the molecule at one or both ends of the molecule, and preferably at least one of the end blocks is also a binding unit, e.g., biotin or a biotin analog.
It is thus an object of the invention to provide arrays having associated chemically modified oligonucleotide and/or polynucleotide polymers to confer substantial nuclease resistance. Nucleases can be used to digest the test substrate binding agent, freeing the associated binding agents for further use. The location of the chemical modification can be determined depending on the binding of the polymer to the substrate and/or the desired nuclease used with the array (e.g., an array to be treated with a 3xe2x80x2 exonuclease can have a 3xe2x80x2 end blocking group on the polymers). The associated oligonucleotides and/or polynucleotides remain unaffected as to their binding capacity with test nucleic acids.
These arrays also offer the significant advantage that the individual chip can be tested for efficacy and/or quality prior to use with a test sample, which is particularly helpful if the amount of test sample is limited or if the array is being used as a medical device and must comply with FDA quality control requirements.
The present invention provides a diagnostic assay using the arrays of the invention to determine the presence of nucleic acids that are indicative of an infectious disease, e.g., viral or bacterial transcripts. The presence of specific nucleic acids indicative of infection is determined by the hybridization pattern of the array after exposure to test samples. The test samples are preferably comprised of RNA, and may be whole cell extracts such as extracts from lymphocytes.
The present invention further provides an assay using the arrays of the invention to determine physiological responses such as gene expression, where the response is determined by the hybridization pattern of the array after exposure to test samples. The test samples are preferably RNA, as the molecules on arrays of the invention show enhanced binding with RNA molecules, although the samples may also be cDNA, whole cell extracts, and the like.
It is an advantage of the associated modified oligonucleotide and/or polynucleotide polymers of the arrays of the invention that the chemical modifications enhance the chemical binding interactions, e.g., increase binding affinity over standard Watson-Crick DNA base pairing with complementary nucleic acids, particularly when binding to mRNA.
It is another advantage of the associated modified oligonucleotide and/or polynucleotide polymers of the arrays of the invention that the polymers have a very high affinity for the substrate.
It is another advantage that the modified oligonucleotide and/or polynucleotide polymers of the array may be synthesized to have approximately the same Tm, by varying the length of the modified polymers. Thus, modified polymers will have the same Tm between compositions allowing for better control of hybridization.
It is another advantage that modified oligonucleotide and/or polynucleotide polymers of the invention hybridize more tightly with complementary RNA sequences than to natural DNA oligonucleotides, allowing the use of shorter binding fragments (e.g., one or more modified polymers in lieu of a complete cDNA).
It is an advantage of the associated modified oligonucleotide and/or polynucleotide polymers of the invention that the acid stable modifications confer an improved stability on the polymers in an acidic environment (e.g., as low as pH of 1 to 2).
It is another advantage of the associated oligonucleotide and/or polynucleotide polymers of the invention that they bind with specificity to test nucleic acids.
It is an object of the invention that the oligonucleotide and/or polynucleotide polymers can be used in a variety of array applications, such as identification of new genes, determination of expression levels, diagnosis of disease, and the like.
These and other objects, advantages, and features of the invention will become apparent to those skilled in the art upon reading the details of the oligonucleotide and/or polynucleotide polymers and uses thereof as more fully described below.